FR2502009A1 - IMPROVEMENTS TO INTERFERON MANUFACTURING PROCESSES - Google Patents

Abstract

THE INVENTION RELATES TO A PERFECTED PROCESS FOR THE PRODUCTION OF INTERFERON. IT CONSISTS OF PRODUCING THE INCUBATION OF A CULTURE OF INTERFERON-PRODUCING CELLS IN THE PRESENCE OF A VITAMINA DERIVATIVE, SUCH AS RETINOIC ACID, PRIOR TO THE INTRODUCTION INTO THE CULTURE OF AN INDUCER OF D SYNTHESIS. 'INTERFERON BY CELLS OF CULTURE.

Description

IMPROVEMENTS TO INTERFERON MANUFACTURING PROCESSES

  Interferon is formed from at least one protein, the synthesis of which by cells is generally induced by a viral infection. It is well known that interferon is capable of inhibiting the development of viruses, whether those which have induced the induction of synthesis or other viruses, even if the inhibition effectiveness of interferon thus synthesized can be variable from one type of virus to another. Interferon synthesized by

  cells is normally excreted in the extracellular medium.

  It can be absorbed by other cells and give them

  thus protect against viral infection.

  Interferon can also, especially when cells

  producers are cultivated in vitro in an environment, preferably

  rence liquid, in principle be recovered from it.

  We know that interferon represents an active ingredient of great therapeutic value, due to its antiviral properties. It can be used successfully, for example for the treatment of herpes virus infections. We also have great hopes

  on the expected ability of interferon to inhibit the development

  development of certain types of cancer.

  The invention therefore relates more particularly to

  improvements made to information production processes

  terferon, or more generally interferons because, as is also well known, there are several forms of interferon depending on the type of producer cells put

  in action. Among the most common producing cells

  mentally used, mention may be made of blood leukocytes,

  lymphocytes, fibroblasts, lymphoblastoid cells, cells

  epithelial cells, for example kidney cells, or HeLa cells, myeloma cells, etc. The invention relates more particularly to the production of interferons which can be used in humans, therefore to interferons produced by cells.

of human origin.

  We know the serious difficulties which

  undermine the effectiveness of known methods of making interferon. Even if we ignore the well-known difficulties encountered in in vitro culture

  of cells of human origin, there is one that pre-

  has a predominant characteristic: these are

  extremely small amounts of interferon which are ex-

  created in the culture media, implemented by

  most of the known methods of producing inter-

  feron. The object of the invention is therefore to remedy the

  less in part to the difficulties encountered

  implementation of known methods, in particular of the type in which an agent is used

  inducer introduced into a cell culture

  ble to produce interferon, since they are likely to synthesize it under the effect of such

inductor.

  The process according to the invention is characterized by bringing these cells into contact, prior to carrying out the induction, with a derivative of retinoic acid, of the type of vitamin A or one of its analogs, hereinafter referred to as a whole under

  the expression "retinoic derivatives".

  Preferably, the retinoic derivative used consists of retinoic acid (3,5-dimethyl (2,6-trimethyl-2,6o-cyclohexane-1 yl 1) -9 acid

  nonatetraene-2,4,6,8-oique) of formula: -

  As examples of other retino- derivatives

  ics likely to be used in the process according to the invention, mention will be made of the counterparts of this retinoic acid, cited on page 35 of the article published by Reuben Lotan in "Biochimica and Biophysica Acta", 605 (1980 ) 33-91, under the title "Effects of vitamin A and its analogs (retinoids) on normal and neoplastic cells" (effects of vitamin A and its analogs

  (retinoids) on normal and neoplastic cells).

  As shown in the table in this

  article, the cyclic terminal group and the poly-

  Many retinoic acids can be subject to numerous modifications or substitutions, the carboxylic group itself being able to be replaced by other groups, in particular alcohols, aldehydes, heteroxides, methyl, lower alkyls, etc. Preferably, recourse is had to those of the derivatives or analogues in question, in which the polyene chain is terminated by a group

  carboxylic, like retinoic acid itself

  same, the formula of which has been indicated above.

  Regarding the interferon producing cells, we can use all lines

  cells, preferably of human origin, suspected

  likely to produce interferon. These cells appear

  advantageously relate to one of the cell lines already identified above. Namalva cell lines, well known to those skilled in the art, such as those derived from cells of patients affected by Burkitt's lymphoma are preferred to date. As

  example of Namalva cells that can be used

  we will mention those called Namalva / WRL which are available at the American Type Culture Collection

(A.T.C.C; CRL 1432).

  With regard to the inducer, recourse may also be had to any type of inductor conventionally used, namely viruses such as the Sendai virus or the NQV virus (Newcastle Disease Virus) which can

  possibly be inactivated, or polynucleo-

  tides or polyribonuelotides, for example poly-

  inosine: polycytidylic (poly I - poly C).

  The discovery that bringing interferon-producing cells into contact with a derivative of the retinoic family or incubating them in the presence of this derivative, before bringing them into contact with an inducer, could lead to a marked increase in the amount of interferon capable of being produced and excreted by said cells in an appropriate culture medium,

  was all the more unexpected as the property was known

  that vitamin A has the ability to inhibit the production of

  terferon by producer cells, when it is

  put in contact with them, before or simultaneously

  nment to contact with an inducer of the type already mentioned, as described by several authors, in particular by JE Blalock and GE Gifford in an article entitled "Inhibition of interferon action by vitamin A" (inhibition of action of interferon by vitamin A), published in "J. Gen. Virol." (1975), 29, 315-324 or in the article by J.E. Blalock and G.E. Gifford entitled "Suppression of interferon production by vitamin A" (Suppression of the production of interferon by the

  vitamin A), published in "J. Gen. Virol." (1976), 32, 143-

147.

  This ability of vitamin A to inhibit the pro-

  duction of cell interferon, before or if

  simultaneously brought into contact with an inductor, is also considered to be acquired knowledge of the state of the art, as evidenced by the extremely complete publication already identified above by Reuben Lotan. Advantageously, the contacting is carried out

  with the culture medium of the cells producing

  terferon, when the level of development of the culture has reached the desired stage. Indeed, we will use a concentration of the derivative of retinoic acid as high

  as possible, compatible with the viability of the cell structure

  milk, preferably nevertheless close to the cytostatic concentration. In this respect, it will be noted that it is naturally possible to have recourse to lower concentrations of retinoic derivatives. The final concentration of the interferon produced in the medium will however be

  lessened. When the cells used belong to

  come from the Namalva line and when the derivative reti-

  no; that used consists of retinoic acid,

  concentrations of the latter will advantageously be adjusted

  tees from 1.10-6 to 1.10 4M. However, this is only an example interval. It is in fact for those skilled in the art to determine the most effective doses, in particular by using a series

  tests with increasing doses allowing to explore

  rer those which will allow, taking into account the type of retino derivative used and the type of producer cells

  of interferon implemented, to establish the zone of con-

most effective centering.

  In particular, we will seek the relative doses of the retinoic agent used, likely to lead to a quasi-interruption of cell growth, without

  however undermine the viability of a proportion

  major tion of the cells then present in the cul-

  ture. Likewise, the contact times between the retino derivative and the culture can be determined by experience. Preferably, these contact times are at least 24 hours, preferably of the order of

48 hours.

  The cells thus treated, previously landed

  of the retino derivative, in particular by washing or any other mode of separation, can then be put

in contact with the inductor.

  For the implementation of the method according to the invention, any conventional culture medium considered suitable for development can be used.

  interferon-producing cells.

  Other characteristics of the invention appear

  will still be during an example of implementation

  preferred of the invention, which is not however given below.

  after that as an additional illustration not limited

  tative of the improved process object of this invention. 1) Culture conditions: Namalva cells are cultured in a non-agitated suspension (unless otherwise indicated) in RPMI 16 culture medium. 40 supplemented with 10% fetal calf serum (RETAHUIN, ARMOR) and antibiotics (CHLORTETRACYCLINE pg / ml, AYiPhOTERICIN B: 1.5 pg / ml) in vials incli-

  born so as to allow oxygenation of the environment. Vo-

  the total lume of the medium thus completed is 400 ml.

  2) Pretreatment with retinoic acid 48 hours before induction, the cells are allowed to sediment by gravity for 6 h at 37 C, the bottle being

straightened vertically.

  Then remove 3/4 of the medium, ie 300 ml, count the cells and add fresh medium to

  have a concentration of O106 cells / ml.

  5.10 5M retinoic acid (SIGMA) is added from a stock solution in ethanol at 10 -2M, to

  - obtain a final concentration of 5.10 5M, i.e. 0.015 mg / ml.

  The bottles are returned to the tilted position and incubated

  at 37 C for 48 hours away from light.

  We stir manually and periodically a few

  seconds, to resuspend the cells.

  3) Induction: After 48 hours, the cells are centrifuged for minutes at 1000 revolutions / minute at 4 C. The supernatant is removed. The cell pellet is resuspended in RPMI medium without

  serum, but containing, in addition to antibiotics, 0.5% (concen-

  tration) of human serum albumin and 10 5M of e-mercapto-

  ethanol, so that the cell concentration is 3.3.106 / ml, a reduction of three times the volume of

02.25009

  initial medium: the cells indeed remained at the concentration of 106 / ml, their multiplication having been

blocked by retinoic acid.

  500 haemagglutinating units (HA units) of Sendai virus (then inactivated by UV irradiation with a germicidal lamp at a dose of 300 ergs / mm 2) are then added per ml of cell suspension. Incubate for approximately 17 hours at 370C in Coràing vials of

  cm2, at the rate of 100 ml per bottle.

  40) Harvest: The suspension is centrifuged for 10 minutes at 2000 revolutions per minute at 41C. The supernatant is brought to pH 2 by addition of 1N HCl and left for three to four days. The solution is then brought back to pH 7 by addition of 4N NaOH. An aliquot is taken for titration: the titles range from 0.5 to 1.2.106 international units / ml

  (international units are defined according to an inter-

  Reference leukocyte feron NIH 6.023.901-527).

  (For comparison, it is indicated that a preparation of interferon carried out under identical conditions, except that the cells used have not, before induction, been pre-incubated in the presence of retinoic acid, led to the same stage of preparation, to a solution titrating

  1.5.104 to 6.104 international units / ml).

  The rest of the supernatant receives trichlora-

  ketic (final concentration 1.5%, from a solution

  at 50%), which precipitates the majority of proteins, including the

  terferon. After 60 minutes at 41C, the precipitate is centrifuged

  t at 10,000 rpm for 15 minutes. The pellet is redissolved in a minimum volume (approximately 1/16 of the initial volume of PBS phosphate buffer (Proc. Nat. Acad. Sci. 1952,

  38, 747), gradually increasing the pH to 7 by addi-

  tion of NaOH 4 N. The PBS used according to the invention does not

  also contains neither calcium nor magnesium. An aliquot is

taken for titration.

  We can optionally dialyze against PBS or

  another buffer for a subsequent purification step

  tion according to a technique known per se.

  Experiments carried out under the same conditions with retinol palmitate (optimal concentration: 5.10-4M) and retinal (optimal concentration: 5.10 5M) gave results identical to those obtained with retinoic acid.

Claims (7)

  1 - Process for the manufacture of interferon by culture of cells producing interferon in a preferably liquid culture medium, and bringing the culture into contact with an interferon inducer, characterized in that the cells of this culture   are, prior to their contacting the information   conductor, brought into contact with a retinoic derivative.   2 - Process according to claim 1, charac-   terized in that the retino derivative is a derivative acid.   3 - Process according to claim 2, charac-   terized in that the acid retinoic derivative is constituted killed by retinoic acid.   4 - Method according to any one of the re-   vendications 1 to 3, characterized in that the concentra-   relative conditions of the culture medium in retinol derivative   that are adjusted to the doses allowing almost   interruption of cell growth, without   both undermine the viability of a proportion   major of the cells then present in the culture.   - Method according to claim 4, characterized in that the contact time between the culture and the retino derivative is at least 24 hours,   preferably around 48 hours.   6 - Process according to claims 3, 4, 5   and 6 taken as a whole, characterized in that the concentration of retinoic acid in the culture medium   is set to a value from 1.iO 6 to 1.10 4M.   7 - Method according to any one of the res-   dications 1 to 6, characterized in that the cells pro-   interferon ductrices used belong to a line of Namalva.   8 - Interferon prepared according to the process   finished in any one of claims 1 to 7.